Disseminated Tumor Cells Research Articles

A high-cholesterol diet promotes the intravasation of breast tumor cells through an LDL–LDLR axis

Most metastases in breast cancer occur via the dissemination of tumor cells through the bloodstream. How tumor cells enter the blood (intravasation) is, however, a poorly understood mechanism at the cellular and molecular levels. Particularly uncharacterized is how intravasation is affected by systemic nutrients. High levels of systemic LDL-cholesterol have been shown to contribute to breast cancer progression and metastasis in various models, but the cellular and molecular mechanisms involved are still undisclosed. Here we show that a high- cholesterol diet promotes intravasation in two mouse models of breast cancer and that this could be reverted by blocking LDL binding to LDLR in tumor cells. Moreover, we show that LDL promotes vascular invasion in vitro and the intercalation of tumor cells with endothelial cells, a phenotypic change resembling vascular mimicry (VM). At the molecular level, LDL increases the expression of SERPINE2, previously shown to be required for both VM and intravasation. Overall, our manuscript unravels novel mechanisms by which systemic hypercholesterolemia may affect the onset of metastatic breast cancer by favouring phenotypic changes in breast cancer cells and increasing intravasation.

It Is What the Surgeon Does Not See That Kills the Patient.

Patients with colon cancer may present at multiple different stages of the disease process. Many patients can be cured of colon cancer as a result of a simple surgical procedure usually performed by minimally invasive techniques. However, there are a variable number of patients, estimated at approximately 10%, who have a more advanced disease. If these patients are treated by the current conventional standard of care, the likelihood for treatment failure is extremely high. These are not patients with known disseminated disease but patients who are at high risk of recurrent disease unless special treatments are initiated preoperatively and intraoperatively. The identification of these patients is by (1) a high-quality CT scan, (2) tumor markers found preoperatively, (3) colonoscopic findings, and (4) symptoms. Patients identified as being at high risk require special preoperative treatments which include neoadjuvant chemotherapy. Intraoperative chemotherapy with HIPEC should occur as part of the treatment if peritoneal metastases are documented by biopsy. In the operating room, a thorough exploration of all possible occult peritoneal spaces for metastatic disease needs to be performed. A modified cytoreductive surgical procedure along with a colon resection is performed in order to minimize sites of occult peritoneal metastases. This includes the greater omentum, ovaries, and tubes in postmenopausal women. Peritonectomy is used to create a shroud around the tumor so that all peritoneum that has been in direct contact with the tumor surface is resected and is used as a barrier against tumor cell dissemination in the process of colon cancer resection. If peritoneal metastases are visualized at any site, HIPEC should be included as part of the treatment package. I am convinced that patients at high risk of recurrence will have an improved outcome with proper preoperative evaluation, preoperative neoadjuvant chemotherapy, and a revised intraoperative management strategy.

Exploring dendritic cell subtypes in cancer immunotherapy: unraveling the role of mature regulatory dendritic cells.

Dendritic cells (DCs) possess a specialized function in presenting antigens and play pivotal roles in both innate and adaptive immune responses. Their ability to cross-present antigens from tumor cells to naïve T cells is instrumental in generating specific T-cell-mediated antitumor responses, crucial for controlling tumor growth and preventing tumor cell dissemination. However, within a tumor immune microenvironment (TIME), the functions of DCs can be significantly compromised. This review focuses on the profile, function, and activation of DCs, leveraging recent studies that reveal insights into their phenotype acquisition, transcriptional state, and functional programs through single-cell RNA sequence (scRNA-seq) analysis. Additionally, the therapeutic potential of DC-mediated tumor antigen sensing in priming antitumor immunity is discussed.

HERC5 downregulation in non-small cell lung cancer is associated with altered energy metabolism and metastasis

BackgroundMetastasis is the leading cause of cancer-related death in non-small cell lung cancer (NSCLC) patients. We previously showed that low HERC5 expression predicts early tumor dissemination and a dismal prognosis in NSCLC patients. Here, we performed functional studies to unravel the mechanism underlying the “metastasis-suppressor” effect of HERC5, with a focus on mitochondrial metabolism pathways.MethodsWe assessed cell proliferation, colony formation potential, anchorage-independent growth, migration, and wound healing in NSCLC cell line models with HERC5 overexpression (OE) or knockout (KO). To study early tumor cell dissemination, we used these cell line models in zebrafish experiments and performed intracardial injections in nude mice. Mass spectrometry (MS) was used to analyze protein changes in whole-cell extracts. Furthermore, electron microscopy (EM) imaging, cellular respiration, glycolytic activity, and lactate production were used to investigate the relationships with mitochondrial energy metabolism pathways.ResultsUsing different in vitro NSCLC cell line models, we showed that NSCLC cells with low HERC5 expression had increased malignant and invasive properties. Furthermore, two different in vivo models in zebrafish and a xenograft mouse model showed increased dissemination and metastasis formation (in particular in the brain). Functional enrichment clustering of MS data revealed an increase in mitochondrial proteins in vitro when HERC5 levels were high. Loss of HERC5 leads to an increased Warburg effect, leading to improved adaptation and survival under prolonged inhibition of oxidative phosphorylation.ConclusionsTaken together, these results indicate that low HERC5 expression increases the metastatic potential of NSCLC in vitro and in vivo. Furthermore, HERC5-induced proteomic changes influence mitochondrial pathways, ultimately leading to alterations in energy metabolism and demonstrating its role as a new potential metastasis suppressor gene.

CLO24-062: Aneuploid Epcam + Disseminated Tumor Cells (DTCs) Correlated With Liver Metastasis and Independently Predict Poor Survival in Small Cell Lung Cancer.

CLO24-062: Aneuploid Epcam + Disseminated Tumor Cells (DTCs) Correlated With Liver Metastasis and Independently Predict Poor Survival in Small Cell Lung Cancer.

Abstract CT223: Dormancy reprograming therapy of 5-azacitidine (AZA) and all-trans retinoic acid (ATRA) in biochemically recurrent prostate cancer

Abstract Microenvironmental signals and epigenetic changes can instruct proliferative disseminated tumor cells to enter dormancy. The combination of Azacitidine (AZA) and All-trans retinoid acid (ATRA) can reprogram active prostate cancer (PCa) cells into a dormant state. Herein, we evaluated the safety and clinical activity of AZA + ATRA in patients (pts) with biochemically recurrent PCa. Eligible pts were those with diagnosis of PCa post-local therapy with rising PSA (PSADT < 10 mo). Pts were excluded if they had radiographic evidence of disease or if they were a candidate for salvage radiation therapy (RT). Eligible pts were randomized 1:1 to arm A vs. B. All pts received Lupron 7.5 mg IM at the start of study. Four weeks later, pts entered a 24-week treatment (tx) phase. Pts in arm A received AZA 40 mg/m2 SQ x 5 days and ATRA 45 mg/m2 PO x 5 days Q4W for 12 weeks (w), followed by 12w of observation. Pts in arm B underwent 12w of observation, followed by 12w of AZA and ATRA at same dosing as arm A. All pts underwent follow up until initiation of next tx. The primary endpoint was safety and tolerability as assessed by CTCAE v5.0. Key secondary endpoints included % of pts with prolongation of PSADT pre- and post-tx and time to next treatment (TTNT). Levels of dormancy biomarkers, BMP4 and BMP7, were quantified using ELISA. Circulating tumor cells (CTCs) were analyzed for positive expression of NR2F1 using immunofluorescence. Wilcoxon signed rank test was used for comparing the PSADT. All statistical analysis was performed in R. Fourteen pts were enrolled (A: N=6, B: N=8) with a median age of 66 (range 54-77). At baseline, mean PSA was 4.75 ng/ml (A: 7.11, B: 2.99) and mean PSADT was 2.89 months (A: 3.51, B: 2.43). Eight (57.1%) pts had a Gleason score > 8 at initial diagnosis (A: 83.3%, B: 37.5%). There were no reported Grade > 3 treatment-related adverse events (TRAEs) and no treatment discontinuations. The most common TRAEs included hot flashes, white blood cell decreased and lymphocyte count decreased (all 8.3% each), and anemia (5.6%). There was prolongation of PSADT pre- and post-tx in 6 (46%) pts, however, overall difference in PSADT was non-significant [p>0.1]. 13 out of 14 (93%) pts had TTNT of > 6 months. One pt developed radiographic progression prior to completion of tx phase and a total of 6 pts (42.8%) within 3 months of tx phase. Out of 8 pts, 37.5% and 75% of pts showed increased levels of BMP4 and BMP7 respectively. Upon tx, CTC count decreased in 3 out of 5 pts analyzed, while CTCs were undetectable in two pts. Upon tx, NR2F1 expression could not be detected due to non-detection of CTCs, except for one pt who showed 100% NR2F1 positive CTCs in follow up. AZA + ATRA appears safe and well tolerated, and there was clinical activity in a small subset of pts potentially correlated with dormancy. Additional prospective clinical studies are needed to elucidate dormancy mechanisms further and optimize dosing strategy. Citation Format: Vaibhav G. Patel, Deepak Singh, Himanshu Joshi, Bobby Liaw, Che-Kai Tsao, Matthew Galsky, Lindsay Diamond, Julio Agguire-Ghiso, William Oh. Dormancy reprograming therapy of 5-azacitidine (AZA) and all-trans retinoic acid (ATRA) in biochemically recurrent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT223.

The Emerging Role of LPA as an Oncometabolite.

Lysophosphatidic acid (LPA) is a phospholipid that displays potent signalling activities that are regulated in both an autocrine and paracrine manner. It can be found both extra- and intracellularly, where it interacts with different receptors to activate signalling pathways that regulate a plethora of cellular processes, including mitosis, proliferation and migration. LPA metabolism is complex, and its biosynthesis and catabolism are under tight control to ensure proper LPA levels in the body. In cancer patient specimens, LPA levels are frequently higher compared to those of healthy individuals and often correlate with poor responses and more aggressive disease. Accordingly, LPA, through promoting cancer cell migration and invasion, enhances the metastasis and dissemination of tumour cells. In this review, we summarise the role of LPA in the regulation of critical aspects of tumour biology and further discuss the available pre-clinical and clinical evidence regarding the feasibility and efficacy of targeting LPA metabolism for effective anticancer therapy.

Vagus nerve signal has an inhibitory influence on the development of peritoneal metastasis in murine gastric cancer

The vagus nerve is the only pathway for transmitting parasympathetic signals between the brain and thoracoabdominal organs, thereby exhibiting anti-inflammatory functions through the cholinergic anti-inflammatory pathway. Despite often being resected during lymph node dissection in upper gastrointestinal cancer surgery, the impact of vagotomy on postoperative outcomes in gastric cancer patients remains unclear. Sub-diaphragmatic vagotomy was performed on C57BL/6 mice. Three weeks later, syngeneic murine gastric cancer cell line YTN16P was injected into the peritoneal cavity, and the number of peritoneal metastases (PM) on the mesentery and omentum compared with control mice. The phenotypes of immune cells in peritoneal lavage and omental milky spots one day after tumor inoculation were analyzed using flow cytometry and immunohistochemistry. Intraperitoneal transfer of 3 × 105 YTN16P significantly increased the number of metastatic nodules on the mesentery in the vagotomy group compared to the control group. The omental metastasis grade was also significantly higher in the vagotomy group. Phenotypic analysis of immune cells in peritoneal lavage did not reveal significant differences after vagotomy. However, vagotomized mice exhibited a notable increase in milky spot area, with a higher presence of cytokeratin(+) tumor cells, F4/80(+) macrophages, and CD3(+) T cells. Vagus nerve signaling appears to regulate the immune response dynamics within milky spots against disseminated tumor cells and inhibits the development of PM. Preserving the vagus nerve may offer advantages in advanced gastric cancer surgery to reduce peritoneal recurrence.

Urolithin A affects cellular migration and modulates matrix metalloproteinase expression in colorectal cancer cells.

Colorectal cancer (CRC) is the world's second most common gastrointestinal malignancy. Preventing tumor cell proliferation and dissemination is critical for patient survival. Polyphenols have a variety of health advantages and can help prevent cancer. The current study examined different cellular activities of the gut-microbiota metabolite urolithin A (UA) on several colon cancer cell lines. The results revealed that UA suppressed cell growth in a dose- and time-dependent manner. In the current investigation, UA substantially affected cell migration in the wound-healing experiment and greatly decreased the number of colonies generated in each CRC cell culture. UA decreased cellular migration in CRC cells 48 h after treatment, which was significant (p < .001) compared to the migration rate in untreated cells. When compared to untreated cells, UA slowed the process of colony formation by reducing the number of colonies or altering their morphological shape. The western blot analysis investigation revealed that UA inhibits cellular metastasis by lowering the expression levels of matrix metalloproteinases 1 and 2 (MMP1 and MMP2) by more than 43% and 41% (p < .001) in HT29, 28% and 149% (p < .001) in SW480, and 90% and 74% (p < .001) in SW620, respectively, at a 100 µM dosage of UA compared to the control. Surprisingly, at a 100 µM dosage of UA, the expression levels of the tissue inhibitor of metalloproteinases 1 (TIMP1) were elevated in HT29, SW480, and SW620 cells treated with 100 µM of UA by more than 89%, 57%, and 29%, respectively. Our findings imply that UA has anticancer properties and might be used therapeutically to treat CRC. The findings provided the first indication of the influence of UA on cellular migration and metastasis in colon cancer cells. All of these data showed that UA might be used as an adjuvant therapy in the treatment of various forms of CRC.

Ultra high content analyses of circulating and tumor associated hybrid cells reveal phenotypic heterogeneity

Persistently high, worldwide mortality from cancer highlights the unresolved challenges of disease surveillance and detection that impact survival. Development of a non-invasive, blood-based biomarker would transform survival from cancer. We demonstrate the functionality of ultra-high content analyses of a newly identified population of tumor cells that are hybrids between neoplastic and immune cells in patient matched tumor and peripheral blood specimens. Using oligonucleotide conjugated antibodies (Ab-oligo) permitting cyclic immunofluorescence (cyCIF), we present analyses of phenotypes among tumor and peripheral blood hybrid cells. Interestingly, the majority of circulating hybrid cell (CHC) subpopulations were not identified in tumor-associated hybrids. These results highlight the efficacy of ultra-high content phenotypic analyses using Ab-oligo based cyCIF applied to both tumor and peripheral blood specimens. The combination of a multiplex phenotypic profiling platform that is gentle enough to analyze blood to detect and evaluate disseminated tumor cells represents a novel approach to exploring novel tumor biology and potential utility for developing the population as a blood-based biomarker in cancer.

Abstract 2966: Senescent cancer cell vaccines induce cytotoxic T cell responses targeting primary tumors and disseminated tumor cells

Abstract Diverse conventional cancer therapies, including genotoxic agents and cell cycle or cell division-targeting drugs, can trigger cancer cell senescence. Senescent cells (SnCs) induced by cancer therapy often display unrepaired chromosomal damage but persist indefinitely. SnCs remain metabolically active and display distinct alterations in transcription, secretion, and membrane protein expression/presentation. While initially viewed as a favorable outcome and likely tumor-suppressive mechanism, therapy-induced senescence has more recently been recognized as contributing to resistance, recurrence and metastasis. Adverse effects may be mediated in part by release of inflammatory mediators as components of the senescence-associated secretory phenotype (SASP). Recent studies have argued for reconsidering the potential benefits of therapy-induced senescence, especially in modulating the tumor microenvironment to potentiate anti-tumor immunity and potentiate immunotherapy. We have explored the potential for SnCs to serve as vaccines, building upon prior findings where we demonstrated the immunogenic senescence triggered by the combination of ionizing radiation (IR) and the PARP1/2 inhibitor veliparib. We find that SnCs are avidly taken up by type I conventional, CD11c+/CD103+ dendritic cells (DCs), promoting their activation and maturation. Thereby, SnCs promote DC function in T cell priming and stimulation. The capacity of SnCs to activate DCs depends on how the SnCs are formed, with our results confirming IR+veliparib induces high immunogenicity and implicating a distinct SASP in these effects. On the other hand, blocking STING signaling limits SnC activation of DCs. In vivo experiments with the murine tumor models CT26 and 4T1 suggest that both SnCs and SnC-pulsed DCs serve as effective anti-tumor vaccines. They not only confer protective immunity against tumor engraftment but also act as therapeutic vaccines, inhibiting both primary and disseminated tumor growth, and synergize with radiotherapy and immune checkpoint blockade. While direct administration of tumor-derived SnCs to patients may raise safety concerns, pulsing autologous DCs with tumor SnCs may help drive anti-tumor immune response, offering a promising path toward personalized cancer immunotherapy. Citation Format: Yue Liu, Joanna Pagacz, Donald J. Wolfgeher, Stephen J. Kron. Senescent cancer cell vaccines induce cytotoxic T cell responses targeting primary tumors and disseminated tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2966.

Abstract 5624: Dec2, a circadian rhythm gene facilitates pancreatic cancer dormancy through evasion of antitumor immunity

Abstract Metastatic recurrence following curative intent oncologic surgery is a major clinical problem in patients with pancreatic ductal adenocarcinoma (PDAC). These recurrences are from disseminated tumor cells (DTCs) and can sometimes occur years after surgery indicating that DTCs undergo a period of dormancy. The mechanisms that drive cancer dormancy and its awakening are poorly understood in part due to a lack of animal models that recapitulate dormant tumor cell biology. We created a novel model of PDAC dormancy where mice undergo surgery to remove the primary tumor and are followed for evidence of metastatic recurrence. Dormant DTCs can be isolated from mice without clinical evidence of recurrence for molecular studies. Single cell RNAseq of dormant DTCs detected that the circadian rhythm gene, Dec2 is upregulated. We knocked out (KO) Dec2 from murine pancreatic cancer cells and when implanted into the model we found almost no mice developed metastatic recurrence. When immunodeficient or immunocompetent mice depleted of CD4 and CD8 T cells were used as hosts, we observed no difference in survival in mice engrafted with Dec WT vs. KO cells indicating the survival benefit in the Dec KO group was due to an immune mediated mechanism. We performed a differential expression analysis on bulk RNAseq data comparing the Dec2 WT vs. KO cells and found antigen presentation a top upregulated pathway. We then observed that surface MHC-I was significantly upregulated in Dec2 KO cells as compared to wild type cells. Further mechanistic studies revealed that proteosome activity was significantly higher in Dec2 KO cells and treatment with proteosome inhibitors decreased surface MHCI levels in Dec2 KO cells. Cut and tag experiments revealed that Dec2 binds the promoter of a number of genes that regulate the proteosome. In conclusion, Dec2 promotes cancer dormancy by down regulating cell surface MHCI which facilitates immune evasion. Citation Format: Lan Wang, Chris Harris, Orjola Prela, Wade C. Narrow, Darren R. Carpizo. Dec2, a circadian rhythm gene facilitates pancreatic cancer dormancy through evasion of antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5624.

Abstract 6718: Evaluating toxicity and efficacy of bispecific EGFRXCD3 T cell engager in solid tumor-bearing humanized animal model

Abstract Background: Cancer immunotherapy has currently become a highly active research area of oncology and a common treatment strategy for several cancer types. One prominent reason is that the utilized animal models could not accurately predict the of toxic response of human immune system to anti-cancer drug. For improving the safety of immune drug development, it is urgent to establish a predictive nonclinical animal platform that can accurately predict human immune response to the drugs and detect potential unwanted toxicity to human patient will be critical. We have demonstrated a disseminated tumor-bearing humanized mouse model that can evaluate the toxicity and efficacy of bispecific T cell engager (BiTE). In this study, we co-engrafted disseminated and solid tumor cells to the same mice and compared with mice engrafted with disseminated tumor or solid tumor. The purpose is to develop a novel tumor-bearing mouse model that can simultaneously evaluate the long-term efficacy against solid tumors in addition to cytokine level assessment. Method: Epidermal growth factor receptor (EGFR) expressing human triple negative breast cancer MDA-MB-231/Luciferase-2A-GFP (MDA) cell line was utilized to establish solid tumors in mice. The cells were implanted into PBMC humanized NSG-MHC class I/II double knock out (DKO) mice via orthotopic injection into mammary fat pad (MFP). Disseminated MDA cells were injected intravenously (IV) and tumor cells normally colonize in mouse lungs. Following EGFRxCD3 BiTE dosing initiation, tumor burden in MFP and lungs were evaluated via in vivo Xenogen imaging system, and solid tumor volume in MFP was measured 2 to 3 times a week by a digital caliper. To evaluate the toxicity in response to EGFRxCD3 BiTE treatment, human inflammatory cytokine levels was analyzed by BD cytometric bead. Clinical observations and body weight change, and human immune cell activation were evaluated. Result: EGFRxCD3 BiTE treatments significantly inhibited MFP solid tumor growth regardless the presence of IV implanted with MDA cells. EGFRxCD3 BiTE treatment induced elevated human inflammatory cytokines from humanized mice implanted with combined MFP and IV tumors. By comparison, the serum cytokine levels were low in mice implanted with MDA cells via MFP alone, suggesting that disseminated tumor cells are required for cytokine production induced by EGFRxCD3 BiTE. Conclusion: Without disseminated tumor, MFP solid tumor-bearing mice can be served as a model for drug efficacy but did not induce inflammatory cytokines by EGFRxCD3 BiTE treatment. Combination of solid and disseminated tumor models in the same mice provide a new approach for simultaneously evaluating the toxic cytokine release and efficacy of immunotherapy drugs. Citation Format: Guoxiang Yang, Li-Chin Yao, James G. Keck, Mingshan Cheng. Evaluating toxicity and efficacy of bispecific EGFRXCD3 T cell engager in solid tumor-bearing humanized animal model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6718.

Abstract 1496: The role of endothelial ACKR1 in breast cancer metastasis

Abstract Background: Tumor cells can co-opt mechanisms of leukocyte extravasation. Atypical Chemokine Receptor 1 (ACKR1) expression in endothelial cells has an established role in promoting leukocyte extravasation; however, the potential for endothelial ACKR1 to facilitate the metastatic process through the extravasation of tumor cells has not been studied. Methods: To understand ACKR1 function, we investigated endothelial ACKR1 expression at the distant metastatic site of the lung, a common site of breast cancer metastasis. We analyzed lung endothelial ACKR1 expression using immunofluorescent staining of lung vasculature during tumor progression in immunocompetent mice bearing orthotopically implanted E0771.LMB breast tumor cells. We assessed the timing of metastatic tumor cell arrival in the lung and evaluated the spatial relationship between disseminated tumor cells and ACKR1-positive vessels. To investigate secreted tumor factors that may mediate ACKR1 regulation in the lung, we performed intratracheal injections of TNF-alpha. We developed an endothelial cell specific ACKR1 conditional knockout mouse model (ACKR1 ECKO) to understand the impact of endothelial ACKR1 on primary tumor growth and subsequent lung metastasis in breast cancer. Results: Our results reveal a significant upregulation of ACKR1 in the lung endothelium of mice implanted with breast cancer cells in the mammary fat pad. Tumor-dependent ACKR1 expression was specifically localized to pulmonary venule endothelium, a common site of leukocyte and tumor cell extravasation. Lung endothelial ACKR1 expression became evident by day 13 after implantation, persisting through endpoint at day 28. Notably, ACKR1 upregulation occurred prior to tumor cell arrival in lungs of tumor bearing mice. We observed spatial clustering of disseminated tumor cells in proximity to ACKR1-positive vessels, implying a preferential site of tumor cell extravasation. In the absence of primary tumors, intratracheal delivery of TNF-alpha induced ACKR1 expression within lungs. When tumors were orthotopically implanted into our ACKR1 ECKO mouse model, we observed a marked decrease in lung metastases compared to control mice. Conclusion/Future Directions: We have determined that pulmonary ACKR1 expression is induced by breast tumors, and vessels expressing ACKR1 appear to act as entry points for lung metastasis. Loss of endothelial ACKR1 reduced metastasis, suggesting that endothelial ACKR1 regulates tumor cell extravasation at distant metastatic sites. We will upregulate lung endothelial ACKR1 expression and directly test its requirement for extravasation using an intravenous tumor cell injection metastasis model. Citation Format: Samuel Tanner Roach, Rishi Patel, Serena Thomas, Chinwe Ewenighi-Amankwah, Joseph Dufraine, L A. Naiche, Jan Kitajewski. The role of endothelial ACKR1 in breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1496.

Abstract 6632: Adipocyte death drives dormant breast cancer cell reactivation

Abstract Despite significant responses to initial treatments, upwards of 30% of breast cancer patients will experience a recurrence of their disease. Unfortunately, there are few clinical features that predict which patients will recur, relegating patients and their doctors to a wait-and-see approach where they must remain vigilant. Recent studies have reported that the presence of disseminated tumor cells (DTCs) in the bone marrow of early-stage breast cancer patients predicts a higher chance of recurrence and that obese patients experience 35%-40% higher rates of recurrence than lean patients. Interestingly, in addition to increased adipocyte size, obese patients experience higher rates of adipocyte apoptosis, macrophage recruitment to adipocyte tissue, and increased inflammation, shown as the formation of crown-like structures (CLSs). Given the rising prevalence of obesity, the need for mechanistic studies to elucidate its contribution to breast cancer dormancy and recurrence is urgent. To investigate how microenvironmental changes associated with obesity affect breast cancer dormancy, we utilized a well-established syngeneic mouse model of ER+ breast cancer dormancy (D2.0R). We found that D2.0R cells remained dormant in the visceral fat of wildtype mice but were reactivated upon adipocyte death due to obesity or genetic manipulation, and this reactivation required the recruitment of macrophages. To understand why macrophages were recruited to the adipose tissue and how they led to dormant tumor cell reactivation, we profiled the microenvironmental changes in visceral fat using single-cell RNA-sequencing and found that the infiltrating macrophages exhibited unique pro-inflammatory and lipid regulatory phenotypes. Further investigation revealed that macrophages activated the integrin-focal adhesion kinase (FAK) pathway in dormant D2.0R cells and the inhibition of integrin and FAK inhibited this activation. Given tumor cells can metastasize to visceral fat depots in breast cancer patients, our findings provide an important model to determine what drives dormant tumor cell growth in adipose tissue. Our study provides novel insights into how tumor microenvironment (TME) impacts breast cancer dormancy and also highlights the importance of adipose tissue as a unique reservoir that potentiates cancer relapse. Citation Format: Xiaoyu Yuan, Qihao Ren, Sheila A. Stewart. Adipocyte death drives dormant breast cancer cell reactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6632.

Abstract 2304: Detection of early-disseminated cancer cells with deep learning-enabled holographic imaging

Abstract Recent research increasingly validates the early dissemination of tumor cells in peripheral blood during the nascent stages of cancer, often preceding clinical identification of the primary tumor. This emerging evidence emphasizes the potential of Circulating Tumor Cells (CTCs) as early indicators for cancer. Despite their significance, CTCs are exceptionally rare compared to normal blood cells, which presents a considerable challenge in detection, particularly during early stages of cancer. Traditional detection methods, such as those based on nucleated cell enrichment, antibody labeling and fluorescence imaging, encounter notable limitations when applied in the context of early detection. These limitations arise from the inherent heterogeneity of CTCs, and the multistep process involved in labeling and manual enumeration, compromising cell viability and resulting in cell loss. This loss becomes particularly problematic in the context of early cancer detection scenarios where the tumor burden is extremely low, and any cell loss can significantly impact assay sensitivity. To overcome these challenges, we propose an innovative CTC detection approach employing a deep learning framework combined with holographic imaging. This technique utilizes coherent light to generate holograms from cell samples, capturing detailed 3D morphological and optical properties of individual cells. We developed a custom convolutional neural network designed to facilitate high-throughput cell sorting in real-time. Trained on healthy blood samples and diverse cancer cell lines, the dataset consists of more than 50 million cellular holograms enhanced by proprietary Gaussian annotation and image augmentation strategies. Moreover, by combining the morphological signatures from holography with antigen signatures (e.g. HER2/ER/PR) in real-time, our model demonstrates the ability to detect less than 1 false positive per 1 million nucleated cells, highlighting its potential as a robust tool for targeted early-stage breast cancer detection. Our technology represents highly sensitive alternative to traditional screening methods, with the promise of enhancing patient outcomes. Citation Format: Nicholas Heller, Kevin Mallery, Nathaniel Bristow, Yash Travadi, Eng Hock Lee, Jiarong Hong. Detection of early-disseminated cancer cells with deep learning-enabled holographic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2304.

Abstract 5535: Intracellular negative regulators of RTK-Ras-ERK signaling alter breast cancer perception of metastatic niche-derived growth factors

Abstract Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that commonly metastasizes to distant organs, such as the lung, resulting in the poor clinical outcomes. TNBC cells commonly overexpress epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) which when bound by microenvironmental growth factors, results in the activation of downstream effector kinases ERK-AKT and initiates transcriptional programs that control cell fate. Tumor cells, however, simultaneously receive many growth factor inputs from their respective microenvironments, many of which stimulate RTKs other than EGFR, that converge at ERK and AKT resulting in different transcriptional and cell fate outputs. This raises questions as to how tumor cells perceive, decode, and respond to a complex set of microenvironmental signals and what the relevance of this process may be for disease pathogenesis and treatment. To this end, our study aimed to determine how disseminated breast cancer cells perceive and respond to a complex growth factor milieu upon seeding and engraftment in the lung. Using live-cell microscopy techniques and TNBC cells carrying biosensors for ERK and Akt signaling pathways, we monitored the dynamic signaling behaviors of single tumor cells, in real-time, as they adapted to the environment in living human lung tissue. Strikingly, we found that individual disseminated tumor cells are less responsive to growth factors once seeded in the lung, as compared to outside this tissue, despite the presence of RTK stimulating ligands. We showed that partial activation of EGFR is necessary for complete activation of ERK signaling by other growth factor-RTK pairs, and expression of downstream ERK target genes, whereas AKT signaling was unaffected. Using quantitative modeling and experimentation, we showed that reshaping of ERK signaling response dynamics occurred via dysregulation of negative regulators that are dominantly controlled by EGFR signaling. Together, these results provide novel insight into how tumor cells perceive and respond to complex microenvironmental signals, which has important implications for drug targeting strategies. Citation Format: Vaibhav Murthy, Cemal Erdem, Jeremy Copperman, Marc Birtwistle, Alexander Davies. Intracellular negative regulators of RTK-Ras-ERK signaling alter breast cancer perception of metastatic niche-derived growth factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5535.

Abstract 198: Selective targeting of endothelial mTORC1 reduces transendothelial fatty acid delivery to improve anti-tumor immunity and suppress metastatic outgrowth

Abstract Tumor metastasis requires sufficient nutrients to support outgrowth of disseminated tumor cells. Cancer cells utilize fatty acids as a key source of energy and biomass, but these nutrients impair the cytotoxic activities of T cells, serving to support metastatic progression. Blood is a rich source of fatty acids, but the vascular endothelium serves as a barrier to limit access to the surrounding tumor tissue. However, it is unclear how long-chain fatty acid delivery to early metastatic tumors is regulated. Here, we report that endothelial mTORC1 promotes transport of fatty acids into developing metastatic tumors, leading to reduced anti-tumor immune responses and improved outgrowth. Using a murine model of metastatic outgrowth in the lung, tumor burden was significantly reduced upon targeted deletion of Raptor, an essential component of the mTORC1 complex, in endothelial cells (RptorECKO). We employed a multifaceted approach using metabolomics, transcriptomics, and molecular assays to identify a Raptor/mTORC1-mediated mechanism of transendothelial fatty acid transport via RAB and CLSTN1 cargo trafficking. In vivo uptake of a fluorescent palmitic acid analog in tumor cells and T cells was reduced in RptorECKO lung metastatic tumors, which correlated with improved markers of cytotoxicity. Combination of low-dose RAD001/everolimus, which selectively inhibits mTORC1 in endothelial cells, with anti-PD1 reduces metastatic disease and impairs long-chain fatty acid uptake in T cells, corresponding to improved anti-tumor immunity. These findings describe a novel mechanism of transendothelial fatty acid transportation during metastatic outgrowth, supporting future development of therapeutic strategies to target endothelial mTORC1 activity. Citation Format: Deanna N. Edwards, Shan Wang, Wenqiang Song, Laura C. Kim, Yoonha Hwang, Jin Chen. Selective targeting of endothelial mTORC1 reduces transendothelial fatty acid delivery to improve anti-tumor immunity and suppress metastatic outgrowth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 198.

Abstract 1386: CD1d-restricted iNKT cells promote liver metastasis from colon cancer

Abstract The liver is a prominent site for metastasis, yet little is known about the role of liver immunity in antimetastatic defense. Compared to other organs, the liver harbors a unique immune environment characterized by the abundance of innate cell lineages, including CD1d-restricted invariant natural killer T (iNKT) cells. To investigate the influence of iNKT cells on metastasis, we established an orthotopic model of colorectal cancer (CRC) with spontaneous dissemination to the liver. We induce the primary tumor by implanting CRC organoids into the colon mucosa of syngeneic C57BL/6 mice, resulting in hepatic macrometastases within 5 weeks. We observed that liver metastasis hardly occurred in mouse strains lacking iNKT cells (Cd1d−/− and Traj18−/− mice), while the growth of the primary tumor was comparable to that in wild-type mice. These results indicate a tumor-promoting function for hepatic iNKT cells. We uncovered that hepatic iNKT cells respond to disseminating cancer by producing IL-4 and IL-13, two cytokines known to activate hepatic stellate cells (HSCs) into fibrogenic myofibroblasts. Indeed, we saw rapid activation of HSCs and deposition of extracellular matrix upon arrival of cancer cells in the liver of wild-type but not iNKT cell-deficient mice. We propose that hepatic iNKT cells contribute to the formation of a supportive niche for disseminated tumor cells by activating HSCs early in the development of hepatic metastasis. Taken together, our findings highlight an essential tumor-promoting role for iNKT cells during the development of liver metastasis and contribute to our understanding of immune regulation of metastasis. Citation Format: Marc Nater, Giulia Lucchiari, Michael Brügger, Tomas Valenta, Virginia Cecconi, Geo Forni, Stephan Benke, Maries van den Broek. CD1d-restricted iNKT cells promote liver metastasis from colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1386.

Abstract 4280: Regulatory T cells contribute to tumor cell dissemination by extracellular matrix remodeling

Abstract Regulatory T (Treg) cells function to enforce peripheral tolerance and are potent suppressors of tumor immunity. In breast cancer, we have shown that Treg cells promote tumor growth by favoring alternative activation of macrophages via suppression of IFN-γ. The tumor-associated extracellular matrix (ECM) is a key regulator of metastatic dissemination and is emerging as a critical regulator of tumor immunity. However, the reciprocal effects of the immune system on the ECM remain elusive. Using murine in vivo, ex vivo and bioengineering models we describe Treg cell-dependent changes in the tumor ECM that facilitate tumor cell movement and metastatic dissemination. Further, Treg cell-dependent matrisome signatures correlate with survival in human breast cancer samples. This work underscores a previously unrecognized role of Treg cells on the ECM that facilitates metastasis and is consistent with tissue Treg cell emergent function as critical regulators of tissue repair and cancer. Citation Format: Ailen Garcia-Santillan, Jessanne Y. Lichtenberg, He Shen, Mikhail G. Dozmorov, Amy L. Olex, Priscilla Y. Hwang, Paula D. Bos. Regulatory T cells contribute to tumor cell dissemination by extracellular matrix remodeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4280.